Introduction: The absence of estrogens in postmenopausal women is linked to an increased risk of type 2 diabetes (T2D), and estradiol replacement can decrease this risk. Notably, exercise can also treat and prevent T2D. This study seeks to understand the molecular mechanisms by which estradiol and exercise induce their beneficial effects via assessing whole-body and cellular changes. Methods: Female Wistar rats were ovariectomized and fed a high-fat diet for 10 weeks and divided into the following 4 experimental groups: 1) no treatment (control), 2) exercise (Ex), 3) estradiol replacement, and 4) Ex+estradiol. Results: Both Ex and estradiol decreased the total body weight gain. However, only exercise effectively reduced the white adipose tissue (WAT) weight gain, food intake, blood glucose levels and serum insulin levels. At the molecular level, exercise increased the non-insulin stimulated pAkt levels in the WAT. In the liver, estradiol increased the protein expression of ACC and FAS, and estradiol decreased the hepatic protein expression of LPL. In the WAT, estradiol and exercise increased the protein expression of ATGL. Conclusion: Exercise provides better protection against T2D when considering whole body measurements, which may be due to increased non-insulin stimulated pAkt in the WAT. However, at the cellular level, several molecular changes in fat metabolism and fat storage occurred in the liver and WAT with estradiol treatment.